EP1813942B1 - Reagenz zur Analyse unreifer Leukozyten und Reagenzkit - Google Patents

Reagenz zur Analyse unreifer Leukozyten und Reagenzkit Download PDF

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EP1813942B1
EP1813942B1 EP07001595A EP07001595A EP1813942B1 EP 1813942 B1 EP1813942 B1 EP 1813942B1 EP 07001595 A EP07001595 A EP 07001595A EP 07001595 A EP07001595 A EP 07001595A EP 1813942 B1 EP1813942 B1 EP 1813942B1
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Prior art keywords
reagent
group
leukocyte
myeloblast
immature
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English (en)
French (fr)
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EP1813942A1 (de
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Tomohiro Tsuji
Ayumu Yoshida
Sinichiro Oguni
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Sysmex Corp
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Sysmex Corp
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5094—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for blood cell populations
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1468—Optical investigation techniques, e.g. flow cytometry with spatial resolution of the texture or inner structure of the particle
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • G01N2015/016—White blood cells
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
    • Y10T436/101666—Particle count or volume standard or control [e.g., platelet count standards, etc.]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
    • Y10T436/107497—Preparation composition [e.g., lysing or precipitation, etc.]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
    • Y10T436/25125—Digestion or removing interfering materials

Definitions

  • the present invention relates to a reagent and a reagent kit for classifying and counting leukocytes contained in a sample taken from a living body.
  • Blood cells are produced in the bone marrow, differentiated from immature cells, grown matured, and migrate to peripheral blood. In healthy adults, immature leukocyte does not appear in peripheral blood, however, immature leukocyte may appear in peripheral blood in patients with leukemia, metastatic bone marrow cancer, severe infectious disease. Therefore, it is extremely important to determine mature leukocytes and immature leukocytes in a biological sample for diagnosis of above-mentioned disorders.
  • reagents for leukocyte determination those disclosed in U.S. Patent Application No. 5958776 are known.
  • a measurement sample in which the reagent and a biological sample are mixed is introduced into a flow cytometer and a light with specific wavelength is irradiated to obtain optical information, it is possible to classify mature leukocytes and immature leukocytes in the specimen based on the optical information and to count them, respectively. Further, it is possible to divide immature leukocytes into myeloblast and immature granulocyte and to count them, respectively.
  • EP0867720 discloses a method for detecting and/or counting an appearance of hematopoietic progenitor cell
  • a reagent containing a nonionic surfactant, sarcosine derivatives (i.e. solubillizing agent) and amino acid and having an osmolarity of 150-600 mOsm/kg.
  • the reagent has an electrical conductivity from 3.0-12.0 mS/cm.
  • EP0525398 discloses a first reagent with a polyoxyethylene-type nonionic surfactant and a second reagent with a dye for staining nucleic acid.
  • the electric conductivity ranges from 5 to 25 mS/cm.
  • EP1542008 discloses a first reagent comprising a nonionic polyoxyethylene-type surfactant, a sarcosine derivative and an amino acid.
  • the second reagent comprises a dye.
  • EP1542008 teaches to adjust the electric conductivity from 6.0-9.0 mS/cm.
  • the present invention provides a reagent for analyzing immature leukocyte contained in a sample, which reagent comprising a surfactant for giving damage to cell membrane of red blood cell and mature leukocyte, a solubilizing agent for causing contraction to damaged blood cell, a sugar, and a dye for staining nucleic acid, wherein said surfactant is a polyoxyethylene-type nonionic surfactant represented by the following chemical formula: R 1 -R 2 -(CH 2 CH 2 O) n -H in the formula, R 1 denotes alkyl group, alkenyl group or -alkynyl group having from 10 to 25 carbon numbers, R 2 denotes -O- or or -COO-, where n is from 10 to 40; wherein said solubilizing agent is at least one selected from the group consisting of sarcosine derivative, salt of sarcosine derivative, cholic acid derivative, and methylglucamide; and wherein said reagent has an o
  • the present invention provides a reagent kit for analyzing immature leukocyte contained in a sample comprising a first reagent which comprises a surfactant for giving damage to cell membrane of red blood cell and mature leukocyte, a solubilizing agent for causing contraction to damaged blood cell, and an osmotic pressure regulator, and a second reagent containing a dye for staining nucleic acid, wherein said surfactant is a polyoxyethylene-type nonionic surfactant represented by the following chemical formula: R 1 -R 2 -(CH 2 CH 2 O) n -H in the formula, R 1 denotes alkyl group, alkenyl group or alkynyl group having from 10 to 25 carbon numbers, R 2 denotes -O- or or -COO-, where n is from 10 to 40; wherein said solubilizing agent is at least one selected from the group consisting of sarcosine derivative, salt of sarcosine derivative, cholic acid derivative, and
  • the present invention provides a reagent kit for analyzing immature leukocyte contained in a sample comprising a first reagent which comprises a surfactant for giving damage to cell membrane of red blood cell and mature leukocyte, a solubilizing agent for causing contraction to damaged blood cell, sugar, and a second reagent containing a dye for staining nucleic acid, wherein said surfactant is a polyoxyethylene-type nonionic surfactant represented by the following chemical formula: R 1 -R 2 -(CH 2 CH 2 O) n -H in the formula, R 1 denotes alkyl group, alkenyl group or alkynyl group having from 10 to 25 carbon numbers, R 2 denotes -O- or or -COO-, where n is from 10 to 40; wherein said solubilizing agent is at least one selected from the group consisting of sarcosine derivative, salt of sarcosine derivative, cholic acid derivative, and methylglucamide; and wherein
  • the present invention it is possible to analyze immature leukocytes and mature leukocytes with good accuracy, and a reagent and a reagent kit for immature leukocytes analysis with broader allowable range of processing conditions of the sample than conventional ones are provided.
  • Nozzle 21 Light source 22: Collimated lens 23: Flow cell 24: Collecting lens 25: Pinhole plate 26: Forward-scattered light detector 27: Collecting lens 28: Dichroic mirror 29: Side-scattered light detector 30: Pinhole plate 31: Side-fluorescence detector 32: Amplifier 33: Amplifier 34: Amplifier 35: Analyzer unit
  • an immature leukocyte analysis reagent (hereafter referred simply to as the reagent) according to the embodiment, it is possible to classify white blood cells contained in a sample into mature leukocytes and immature leukocytes and to count them, respectively. It is also possible to divide mature leukocytes into lymphocytes, monocytes and granulocytes and count them, respectively. Especially when the reagent is used, it is possible to further classify immature leukocytes into immature granulocytes and myeloblasts and to count them with good accuracy.
  • Immature leukocyte as used herein means immature white blood cell which is not present in the peripheral blood of healthy individuals, but is present in the bone marrow.
  • myeloblast promyelocyte, medullocell, metamyelocyte or the like are mentioned. Promyelocyte, medullocell, metamyelocyte are sometimes referred to as the immature granulocyte.
  • Myeloblast also includes hematopoietic precursor cell of white blood cell system such as bone marrow stem cell (CFU-GEMN), neutrophil, macrophage colony forming cell (CFU-GM), eosinophil colony forming cell (CFU-EOS) or the like.
  • CFU-GEMN bone marrow stem cell
  • CFU-GM macrophage colony forming cell
  • CFU-EOS eosinophil colony forming cell
  • sample for biological sample to be used for the measurement, there is no particular restriction as long as the sample includes white blood cell; and blood, urine, bone marrow aspirate, and sample taken by apheresis may be exemplified.
  • the reagent according to the embodiment includes surfactant as defined in claim 1 that gives damage to cell membrane of red blood cell and mature leukocyte, solubilizing agent as defined in claim 1 that causes damaged blood cell to constrict and nucleic acid staining dye.
  • the reagent has an osmotic pressure, an electric conductivity and a concentration of sodium chloride as defined in claim 1.
  • this surfactant gives damage to cell membrane of red blood cell and mature leukocyte, it does not give substantial damage to cell membrane of immature leukocyte. Damaged blood cell of red blood cell, mature leukocyte or the like cause constriction by the action of solubilizing agent.
  • polyoxyethylene-type nonionic surfactants having the following chemical formula are used: R 1 -R 2 -(CH 2 CH 2 O) n -H
  • R 1 denotes alkyl group, alkenyl group or alkynyl group having from 10 to 25 carbon numbers
  • R 2 denotes -O- or or -COO-, where n is from 10 to 40.
  • polyoxyethylene (16) oleyl ether polyoxyethylene (20) lauryl ether, polyoxyethylene (15) oleyl ether or the like.
  • Preferable concentration of the surfactant to be contained by the reagent is depending on types of the surfactant.
  • polyoxyethylene (16) oleyl ether for example, from 1000 to 50000 ppm is preferable, from 10000 to 35000 ppm is more preferable.
  • the surfactant may be used alone or more than two types of surfactants may be used together.
  • solubilizing agent sarcosine derivative or salt thereof, cholic acid derivative, methylglucamide or the like are used.
  • Sarcosine derivative has the following chemical formula: (In the formula, R 1 denotes alkyl group having from C10 to 22, and n is from 1 to 5.)
  • Cholic acid derivative has the following chemical formula: (In the formula, R 1 is hydrogen atom or hydroxyl group.)
  • Methylglucamide has the following chemical formula: (In the formula, n is from 5 to 7.)
  • sarcosine derivative or salt thereof When sarcosine derivative or salt thereof is used, its concentration in the reagent is preferably from 200 to 3000 ppm. When cholic acid derivative is used, its concentration is preferably from 100 to 1000 ppm. When methylglucamide is used, its concentration is preferably from 1000 to 8000 ppm.
  • sarcosine derivative or salt thereof N-lauroyl sarcosine sodium, lauroyl methyl ⁇ -alanine sodium, lauroyl sarcosine or the like are mentioned.
  • cholic acid derivative or salt thereof CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate), CHAPSO ([3-cholamidopropyl] dimethylammonio)-2-hydroxy-1-propane sulfonate) or the like are mentioned.
  • methylglucamide As for concrete example of methylglucamide, MEGA8 (octanoyl-N-methylglucamide), MEGA9 (nonanoyl-N-methylglucamide), MEGA10 (decanoyl-N-methylglucamide) or the like are mentioned.
  • the dye there is no particular restriction as long as it specifically stains nucleic acid, while fluorescent dye is preferred.
  • red blood cell without nucleus is hardly stained, while white blood cell with nucleus is strongly stained.
  • white blood cell with nucleus is strongly stained.
  • staining strength it is possible to discriminate red blood cell and white blood cell.
  • mature leukocyte having cell membrane heavily damaged as to allow transmission of dyes is strongly stained, while immature leukocyte is hardly stained.
  • the type of dye is selected appropriately depending on the light being irradiated.
  • dyes having the following chemical formula (wherein R 1I is hydrogen atom or a lower alkyl group; R 2I and R 3I I are independently hydrogen atom, a lower alkyl group or a lower alkoxy groups; R 4I is hydrogen atom, an acyl group or a lower alkyl group; R 5I is hydrogen atom or an optionally substituted lower alkyl group; Z is sulfur atom, oxygen atom or carbon atom substituted with a lower alkyl group; n is 1 or 2; and X I- is an anion.)
  • lower alkyl group in R 1I is straight chain or branched chain alkyl group having from 1 to 6 carbon numbers.
  • methyl group, ethyl group, propyl group, butyl group, isobutyl group, sec-butyl group, ter-butyl group, pentyl group, hexyl group or the like are mentioned, and of them, methyl group and ethyl group are preferable.
  • R 2I and R 3I I are the same as above, and as for lower alkoxy group, alkoxy having from 1 to 6 carbon numbers is meant.
  • methoxy group, ethoxyl group, propoxy group or the like are mentioned, while of them, methoxy group and ethoxyl group are preferable.
  • R 2I and R 3I I are preferably hydrogen atom.
  • acyl group in R 4I acyl group derived from aliphatic carboxylic acid is preferable. Specifically, acetyl group, propionyl group or the like are mentioned, and of them, acetyl group is preferable. Further, lower alkyl group is similar to the above.
  • Lower alkyl group in R 5I is similar to the above, and lower alkyl group that may be substituted denotes lower alkyl group that may be substituted with from 1 to 3 hydroxy groups, halogen atom (fluorine, chlorine, bromine or iodine) or the like. Of them, methyl group and ethyl group substituted with one hydroxy group is preferable.
  • Lower alkyl group in Z is similar to the above, and as for Z, sulfur atom is preferable.
  • halogen ion fluorine, chlorine, bromine or iodine ion
  • boron halogenide ion BF4-, BC14-, BBr4-or the like
  • phosphide compound ion halogen oxyacid ion, fluorosulfuric acid ion, methylsulfuric acid ion, tetraphenyl borate compound ion having alkyl group having aromatic ring halogen or halogen as the substitution group, or the like are mentioned.
  • bromine ion or BF4- is preferable.
  • the concentration of above-mentioned dyes in the reagent is preferably from 0.01 to 500 ppm, more preferably from 0.1 to 200 ppm.
  • the dye may be used alone or more than two types of dyes may be used together.
  • the osmotic pressure of the reagent is adjusted to from 150 to 600 mOsm/kg.
  • the reagent contains sugar. Even if reaction time is lengthened or reaction temperature is increased by adjustment of the osmotic pressure of the reagent using sugar, myeloblast is hardly damaged, and it is possible to classify myeloblast and mature leukocyte and to count them accurately.
  • sodium chloride may be also contained in the reagent in concentrations as defined in claim 1.
  • concentration of sodium chloride in the reagent is in concentrations as defined in claim 1 preferably from 0.01 to 3 g/L, and 0 g/L (not contained) is more preferable.
  • sugar to be contained in the reagent is not limited in particular, monosaccharide, polysaccharide, sugar alcohol or the like may be used.
  • monosaccharide glucose, fructose or the like are exemplified; for polysaccharide, arabionose or the like are exemplified; for sugar alcohol, xylitol, sorbitol, mannitol, ribitol are exemplified.
  • Sugar concentration in the reagent is preferably from 10 to 75 g/L, more preferably from 20 to 50 g/L. Of these sugars, one type may be used or more than two types may be used together.
  • buffering agent In order to adjust pH of the reagent, it is preferable to add buffering agent to the reagent.
  • buffering agent Good' buffer such as HEPES, phosphoric acid buffering agent, or the like may be used, and pH osmotic pressure regulator such as sodium hydroxide may be used. It is preferable that pH of the reagent is adjusted to from 5.0 to 9.0.
  • the reagent kit includes a first reagent containing a surfactant as defined in claim 10 to give damage to cell membrane of red blood cell and mature leukocyte, a solubilizing agent as defined in claim 10 to cause constriction to damaged blood cells, sugar and a second reagent containing dyes.
  • the first reagent has an osmotic pressure, an electric conductivity and a concentration of sodium chloride as defined in claim 10.
  • dyes in the second reagent are preferably being dissolved in the organic solvent.
  • a reagent kit includes a first reagent containing a surfactant as defined in claim 10 to give damage to cell membrane of red blood cell and mature leukocyte, a solubilizing agent as defined in claim 10 to cause constriction to damaged blood cells, osmotic pressure regulator; and a second reagent containing nucleic acid staining dye.
  • the first reagent has an osmotic pressure, an electric conductivity and a concentration of sodium chloride as defined in claim 10.
  • the osmotic pressure regulator is added to allow adjustment of osmotic pressure and electric conductivity of the first reagent to a level as defined in claim 10.
  • the osmotic pressure of the first reagent is adjusted to from 150 to 600 mOsm/kg.
  • the electric conductivity of the first reagent is adjusted to from 0.1 to 2 ms/cm.
  • sugar, amino acid or the like may be used.
  • amino acid valine, proline, glycine, alanine or the like may be used, while it is preferable to use either of glycine or alanine, or both.
  • concentration of amino acid from 1 to 50 g/L is preferable, and from 10 to 30 g/L is more preferable.
  • sodium chloride is further added to the reagent as the osmotic pressure regulator, its concentration in the reagent is as defined in claim 10 preferably from 0.01 to 3 g/L, and 0 g/L is more preferable, so that, as mentioned previously, measurements of myeloblast may not be affected.
  • Mixing ratio of the biological sample and reagent is preferably from 1:10 to 1 : 1000.
  • Reaction of blood cell in the biological sample with the reagent is preferably carried out for from 3 to 15 sec at from 20 to 40°C. When the reaction temperature is high, reaction time may be shortened, and when reaction temperature is low, reaction time may be lengthened.
  • a light is irradiated to blood cells in the measurement sample flowing through the flow cell to acquire optical information such as scattering light and fluorescence or the like, and type of blood cell is identified based on this information.
  • FIG. 1 a flow cytometer as shown in FIG. 1 may be used.
  • measurement of myeloblast will be explained hereafter in detail referring to FIG. 1 .
  • a measurement sample discharged from a nozzle 6 flows through an orifice part of a flow cell 23.
  • blood cells in the sample pass through the orifice part in line.
  • a light emitted from a light source 21 is irradiated via a collimated lens 22 to blood cells flowing through the flow cell 23.
  • side-scattered light, side-fluorescence and forward-scattered light are generated.
  • Side-scattered light is incident to a side-scattered light detector (photomultiplier tube) 29 via a collecting lens 27 and a dichroic mirror 28.
  • Forward-scattered light signal being output from the forward-scattered light detector 26 side-scattered light signal output from the side-scattered light detector 29, and side-fluorescence signal output from the side-fluorescence detector 31 are amplified respectively by an amplifier 32, an amplifier 33 and an amplifier 34, and enter into an analyzer unit 35.
  • the analyzer unit 35 calculates forward-scattered light intensity, side-scattered light intensity and fluorescence intensity from the forward-scattered light signal, side-scattered light signal and side-fluorescence signal received.
  • the analyzer unit 35 generates a first two-dimensional distribution chart based on two axes of forward-scattered light intensity and fluorescence intensity and identifies on this two-dimensional chart a region where total leukocyte in the sample appear (total leukocyte region) . Further, it generates a second two-dimensional distribution chart based on two axes of side-scattered light intensity and fluorescence intensity for cells appearing on the total leukocyte region.
  • a region where mature leukocytes appear (mature leukocyte region), a region where lymphocytes appear (lymphocyte region), a region where monocytes appear (monocyte region), and a region where granulocytes appear (granulocyte region) are set. Further, a region where myeloblasts appear (myeloblast region) and a region where immature granulocytes appear (immature granulocyte region) are identified. The number of cells appearing on the myeloblast region is counted as the number of myeloblasts contained in the sample, and the number of cells appearing on the immature granulocyte region is counted as the number of immature granulocyte contained in the sample.
  • myeloblast is small in cell size and is of single nucleus, and therefore, forward-scattered light intensity is strong and side-scattered light intensity is weak.
  • myeloblast is hardly stained, and therefore, its fluorescence intensity is weak. Immature granulocyte is large in cell size and its nucleus is segmented, and therefore, its forward-scattered light intensity and side-scattered light intensity are weak. Further, since it is hardly stained as mentioned before, its fluorescence intensity is weak.
  • a first reagent A and a second reagent with the following compositions were prepared:
  • Osmotic pressure of the first reagent A was 280 mOsm/kg and electric conductivity was 0.59 mS/cm.
  • a first two-dimensional distribution chart based on two axes of side-scattered light intensity and fluorescent intensity obtained was prepared and total leukocyte region was identified. This is shown in FIG. 2 . The number of cells appeared in the total leukocyte region was counted as the total leukocyte count.
  • a second two-dimensional distribution chart based on two axes of side-scattered light intensity and fluorescence intensity was prepared, and a region where side-scattered light intensity was low and fluorescence intensity was low was identified as the myeloblast region. This is shown in FIG. 3 .
  • the number of cells appearing on the myeloblast region was counted as the number of myeloblasts.
  • Myeloblast ratio Number of myeloblast/Total leukocyte count ⁇ 100
  • Myeloblast ratio was calculated similarly as Example 1 except that a reagent and blood sample A were reacted at 35°C. Myeloblast ratio was 42.6%.
  • the first two-dimensional distribution chart prepared in Example 2 is shown in FIG. 4 and the second two-dimensional distribution chart is shown in FIG. 5 .
  • Myeloblast ratio was calculated similarly as Example 1 except that 1 mL of reagent containing polyoxyethylene (16) oleyl ether 24.0 g/L, N-lauroyl sarcosine sodium 1.5 g/L, DL-methionine 20.0 g/L, HEPES 12.0 g/L, 1N-NaOH 0.3 g/L, NaCl 4.0 g/L and dye A 3.0 mg/L, and 33 ⁇ L of blood sample B were mixed and reacted. Myeloblast ratio was 25.1 %.
  • the first two-dimensional distribution chart prepared in Comparison Example 1 is shown in FIG. 6 and the second two-dimensional distribution chart is show in FIG. 7 .
  • Osmotic pressure of this reagent was 350 mOsm/kg and electric conductivity was 7.4 mS/cm.
  • Myeloblast ratio was calculated similarly as Comparison Example 1 except that a reagent and blood sample B were reacted at 35°C. Myeloblast ratio was 7.2%. Meanwhile, the first two-dimensional distribution chart prepared in Comparison Example 2 is shown in FIG. 8 and the second two-dimensional distribution chart is shown in FIG. 9 .
  • a first reagent B was prepared similarly as the first reagent A except that xylitol 39.56 g/L in lieu of arabinose, polyoxyethylene (16) oleyl ether 25000 ppm in lieu of 20000 ppm, N-lauroyl sarcosine sodium 750 ppm in lieu of 500 ppm were caused to be contained.
  • Osmotic pressure of the first reagent B was 280 mOsm/kg and electric conductivity was 0.59 mS/cm.
  • Myeloblast ratio was calculated similarly as Example 1 except that first reagent B was used in lieu of first reagent A, and blood sample C was used in lieu of blood sample A. Further, myeloblast ratio was calculated similarly as Example 1 except that first reagent B was used in lieu of first reagent A, blood sample C was used in lieu of blood sample A, and the reagent was reacted with blood sample C for 12 sec in lieu of 7 sec.
  • a first reagent C was prepared similarly as the first reagent B except that arabinose 39.52 g/L in lieu of xylitol was caused to be contained. Osmotic pressure of the first reagent C was 280 mOsm/kg and electric conductivity was 0.62 mS/cm.
  • Myeloblast ratio was calculated similarly as Example 3 except that first reagent C was used in lieu of first reagent B.
  • a first reagent D was prepared similarly as the first reagent B except that alanine 23.16 g/L in lieu of xylitol was caused to be contained. Osmotic pressure of the first reagent D was 280 mOsm/kg and electric conductivity was 0.61 mS/cm.
  • Myeloblast ratio was calculated similarly as Example 3 except that first reagent D was used in lieu of first reagent B.
  • a first reagent E was prepared similarly as the first reagent B except that glycine 19.52 g/L in lieu of xylitol was caused to be contained. Osmotic pressure of the first reagent E was 280 mOsm/kg and electric conductivity was 0.63 mS/cm.
  • Myeloblast ratio was calculated similarly as Example 3 except that first reagent E was used in lieu of first reagent B.
  • Myeloblast ratio was calculated similarly as Example 1 except that blood sample C was used in lieu of blood sample A. Further, myeloblast ratio was calculated similarly as Example 1 except that blood sample D was used in lieu of blood sample A, and the reagent was reacted with blood sample C for 12 sec in lieu of 7 sec.
  • FIG. 10 is a graph showing how much is reduced myeloblast ratio when the sample and reagent are reacted for 12 sec compared to myeloblast ratio when reacted for seven sec.
  • a first reagent F containing the following compositions was prepared:
  • Myeloblast ratio was calculated similarly as Example 1 except that the first reagent F was used in lieu of the first reagent A and the blood sample D was used in lieu of blood sample A. Myeloblast ratio was 45%.
  • the first two-dimensional distribution chart drawn in Example 7 is shown in FIG. 11 and the second two-dimensional distribution chart is shown in FIG. 12 . Mature leukocyte region where mature leukocyte appears was also set to the second two-dimensional distribution chart shown in FIG. 12 .
  • myeloblast ratio of the blood sample D calculated using a microscope was 58.5%. From the fact that myeloblast ratio measured in Example 7 and myeloblast ratio calculated using the microscope showed approximation, it has been confirmed that when a reagent of Example 7 is used, mature leukocyte and myeloblast in the blood sample can be identified accurately and myeloblast can be counted accurately.
  • Myeloblast ratio was calculated similarly as Example 5 except that a blood sample G was used in lieu of the blood sample D. Myeloblast ratio was 5.5%.
  • the first two-dimensional distribution chart drawn in Example 8 is shown in FIG. 13 and second two-dimensional distribution chart is shown in FIG. 14 .
  • myeloblast ratio of the blood sample E calculated using the microscope was 6.3%. From the fact that myeloblast ratio measured in Example 8 and myeloblast ratio calculated using the microscope showed approximation, it has been confirmed that when a reagent of Example 8 is used, mature leukocyte and myeloblast in the blood sample can be identified accurately and myeloblast can be counted accurately.
  • immature granulocyte region where immature granulocyte appears was set to the first two-dimensional distribution chart shown in FIG. 13 , and the number of cells appearing in this region was counted as the number of immature granulocyte. Based on this value, ratio of immature granulocyte to total leukocyte count (immature granulocyte ratio) was calculated. Immature granulocyte ratio was 10%.
  • immature granulocyte ratio of the blood sample E calculated using the microscope was 13%. From the fact that immature granulocyte ratio measured in Example 8 and immature granulocyte ratio calculated using the microscope showed approximation, it has been confirmed that when the reagent of Example 8 is used, mature leukocyte, myeloblast and immature granulocyte in the blood sample can be identified accurately and immature granulocyte as well as myeloblast can also be counted accurately.
  • Immature granulocyte ratio was calculated similarly as Example 5 except that the blood sample F was used in lieu of blood sample D. Immature granulocyte ratio was 12.4%. Meanwhile, the first two-dimensional distribution chart drawn in Example 9 is shown in FIG. 15 and second two-dimensional distribution chart is shown in FIG. 16 .

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Claims (10)

  1. Reagens zum Untersuchen unreifer Leukozyten, die in einer Probe enthalten sind, wobei das Reagens umfasst:
    ein Tensid, um der Zellmembran von roten Blutzellen und reifen Leukozyten Schaden zuzufügen, ein Solubilisierungsmittel, um Zusammenziehen der geschädigten Blutzelle zu verursachen, einen Zucker und eine Farbe zum Färben von Nukleinsäure,
    wobei das Tensid ein Polyoxyethylen-Typ nicht-ionisches Tensid ist, das durch die folgende chemische Formel wiedergegeben wird:

            R1-R2-(CH2CH2O)n-H

    in der Formel bezeichnet R1 eine Alkylgruppe, Alkenylgruppe oder Alkinylgruppe mit einer Kohlenstoffzahl von 10 bis 25,
    R2 bezeichnet -O- oder
    Figure imgb0015
    oder -COO-, wobei n von 10 bis 40 ist;
    wobei das Solubilisierungsmittel mindestens eines ausgewählt aus der Gruppe bestehend aus einem Sarcosinderivat, Salz eines Sarcosinderivats, Cholsäurederivat und Methylglucamid ist; und
    wobei das Reagens einen osmotischen Druck von 150 bis 600 mOsm/kg und eine elektrische Leitfähigkeit von 0,1 bis 2 mS/cm hat; und
    wobei die Konzentration von Natriumchlorid im Reagens weniger als 3 g/l ist.
  2. Reagens gemäß Anspruch 1, wobei der Zucker mindestens einer ausgewählt aus der Gruppe bestehend aus Xylitol, Arabinose, Glucose, Mannitol, Sorbitol, Ribitol ist.
  3. Reagens gemäß Anspruch 1 oder 2, das von 10 bis 75 g/l des Zuckers enthält.
  4. Reagens gemäß einem beliebigen der Ansprüche 1 bis 3, das im wesentlichen kein Natriumchlorid enthält.
  5. Reagens gemäß einem beliebigen der Ansprüche 1 bis 4, dessen pH von 5,0 bis 9,0 ist.
  6. Reagenskit zum Untersuchen unreifer Leukozyten, die in einer Probe enthalten sind, umfassend ein erstes Reagens, das umfasst: ein Tensid, zum Verursachen von Schaden an der Zellmembran von roten Blutzellen und reifen Leukozyten, ein Solubilisierungsmittel, zum Verursachen des Zusammenziehens der geschädigten Blutzelle und einen Regulator des osmotischen Drucks, sowie ein zweites Reagens, das eine Farbe zum Färben von Nukleinsäure enthält;
    wobei das Tensid ein Polyoxyethylen-Typ nicht-ionisches Tensid ist, das durch die folgende chemische Formel wiedergegeben wird:

            R1-R2-(CH2CH2O)n-H

    in der Formel bezeichnet R1 eine Alkylgruppe, Alkenylgruppe oder Alkinylgruppe mit einer Kohlenstoffzahl von 10 bis 25,
    R2 bezeichnet -O- oder
    Figure imgb0016
     oder -COO-, wobei n von 10 bis 40 ist;
    wobei das Solubilisierungsmittel mindestens eines ausgewählt aus der Gruppe bestehend aus einem Sarcosinderivat, Salz eines Sarcosinderivats, Cholsäurederivat und Methylglucamid ist; und
    wobei das Reagens einen osmotischen Druck von 150 bis 600 mOsm/kg und eine elektrische Leitfähigkeit von 0,1 bis 2 mS/cm hat; und
    wobei die Konzentration von Natriumchlorid im Reagens weniger als 3 g/l ist.
  7. Reagenskit gemäß Anspruch 6, wobei der Regulator des osmotischen Drucks mindestens einer ausgewählt aus der Gruppe bestehend aus Zucker und Aminosäure ist.
  8. Reagenskit gemäß Anspruch 7, wobei die Aminosäure mindestens eine ausgewählt aus der Gruppe bestehend aus Glycin und Alanin ist.
  9. Reagenskit gemäß Anspruch 7 oder 8, der von 1 bis 50 g/l der Aminosäure enthält.
  10. Reagenskit zum Untersuchen unreifer Leukozyten, die in einer Probe enthalten sind, umfassend ein erstes Reagens, das umfasst: ein Tensid, zum Verursachen von Schaden an der Zellmembran von roten Blutzellen und reifen Leukozyten, ein Solubilisierungsmittel, zum Verursachen des Zusammenziehens der geschädigten Blutzelle und Zucker, sowie ein zweites Reagens, das eine Farbe zum Färben von Nukleinsäure enthält;
    wobei das Tensid ein Polyoxyethylen-Typ nicht-ionisches Tensid ist, das durch die folgende chemische Formel wiedergegeben wird:

            R1-R2-(CH2CH2O)n-H

    in der Formel bezeichnet R1 eine Alkylgruppe, Alkenylgruppe oder Alkinylgruppe mit einer Kohlenstoffzahl von 10 bis 25,
    R2 bezeichnet -O- oder
    Figure imgb0017
     oder -COO-, wobei n von 10 bis 40 ist;
    wobei das Solubilisierungsmittel mindestens eines ausgewählt aus der Gruppe bestehend aus einem Sarcosinderivat, Salz eines Sarcosinderivats, Cholsäurederivat und Methylglucamid ist; und
    wobei das Reagens einen osmotischen Druck von 150 bis 600 mOsm/kg und eine elektrische Leitfähigkeit von 0,1 bis 2 mS/cm hat; und
    wobei die Konzentration von Natriumchlorid im Reagens weniger als 3 g/l ist.
EP07001595A 2006-01-27 2007-01-25 Reagenz zur Analyse unreifer Leukozyten und Reagenzkit Active EP1813942B1 (de)

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